WO1996021010A1

WO1996021010A1 - Male sterile brassica oleracea plants

Info

Publication number: WO1996021010A1
Application number: PCT/US1995/006265
Authority: WO
Inventors: Maury L. Boeshore; Rosaline Z. Deng; Glen E. Ruttencutter; John F. Reynolds
Original assignee: Asgrow Seed Company
Priority date: 1994-12-30
Filing date: 1995-06-07
Publication date: 1996-07-11
Also published as: AU2761495A; IL116112A0

Abstract

A cytoplasmic male sterile, cold tolerant, diploid Brassica oleracea plant is provided. The plant includes chloroplasts of fertile Brassica oleracea and a recombined mitochondrial genome provided by Ogura CMS cytoplasm and the fertile Brassica oleracea plant.

Description

TITLE

MALE STERILE BRASSICA OLERACEA PLANTS BACKGROUND OF THE INVENTION

This invention concerns the development of new parental lines of Brassica oleracea . The parental lines are used to produce hybrid seed. Specificallv, this invention enables a plant breeder to incorporate the desirable qualities of cytoplasmic male sterility (CMS) and cold tolerance into a commercially desirable hybrid variety of Brassica oleracea . Male sterility is of value in Brassica oleracea hybrid seed breeding because normal flowers are self- pollinating. Male sterile lines do not produce viable pollen and cannot self-pollinate. By eliminating the pollen of one parental variety in a cross, a plant breeder is assured of obtaining hybrid seed of uniform quality. Cytoplasmic male sterility (CMS) has not generally been readily available in Brassica oleracea varieties. Thus, commercial producers of hybrid seed use nuclear self-incompatibility systems to avoid self-pollination during seed production.

Unfortunately, this system is not 100% effective, which results in impure hybrid seed lots. Also, it is time consuming, laborious and thus costly to introduce this genetic system into all breeding lines.

In Raphanus sativus, a cytoplasm was discovered that confers male sterility. This cytoplasm is known as Ogura CMS cytoplasm and the DNA from the mitochondria and chloroplasts contained in the cytoplasm is

genotypically different from the DNA in the cytoplasm of fertile Brassica oleracea plants. The Ogura type CMS cytoplasm can be introduced into Brassica oleracea inbred lines by repeated backcrosses. In the resulting Ogura CMS Brassica oleracea plants, the mitochondria of Raphanus sativus result in a CMS phenotype. The presence of the chloroplasts of Raphanus sativus, however, results in chlorosis, i.e., chlorophyll deficiency, when the plants are grown at low

temperature, which consequently results in yield losses. This makes this type of Ogura CMS Brassica oleracea plant of little use in commercial hybrid seed production.

U.S. Patent 5,254,802 to Hoekstra et al . reports the production of Brassica oleracea plants having

mitochondria of the Ogura CMS cytoplasm and cold tolerant chloroplasts of normal fertile Brassica oleracea . Hoekstra et al. report that their plants have cytoplasmic male sterility and do not show

chlorosis when grown at low temperature. Hoekstra et al . report that they obtained these Ogura CMS, cold tolerant Brassica oleracea plants by fusion of Brassica oleracea protoplasts having commercially desirable nuclear traits with inactivated or nucleus-free

f protoplasts of an Ogura CMS Brassica oleracea plant, followed by regeneration into plants of the thus obtained allogenic cells. In the '802 patent and its accompanying file history, Hoekstra et al. appear to posture their invention as Brassica oleracea plants having the whole mitochondrial genome of the Ogura CMS cytoplasm and cold tolerant Brassica chloroplasts. Specifically, they appear to emphasize that they have achieved cellular organelle segregation without organelle genome recombination (whole Ogura CMS mitochondria coupled with fertile Brassica oleracea chloroplasts). Thus, a need exists for a method of protoplast fusion in Brassica oleracea that allows for the random production of recombined mitochondrial genomes having unique genotypic

characteristics. SUMMARY OF THE INVENTION

The present invention provides diploid Brassica

oleracea plants, such as, broccoli, cabbage,

cauliflower, brussel sprouts, kale, and kohlrabi, etc., that have cytoplasmic male sterility (CMS) and do not show chlorosis when grown at low temperatures. That is, these plants are cold tolerant. These plants include cells comprising chloroplasts derived from a fertile Brassica oleracea plant and a recombined mitochondrial genome. These plants show good seed set and are thus capable of producing commercially

acceptable amounts of hybrid seeds. Preferably, the plants of the present invention are broccoli and cabbage plants. The present invention also provides seeds produced from these plants that can transmit these characteristics, and the isolated and purified mitochondria from these plants. As used herein, the terms "isolated and purified" refer to in vi tro

isolation of a mitochondrion from its natural cellular environment.

The invention is based on the finding that these randomly produced recombined mitochondria contain portions of the Ogura CMS mitochondria and the fertile Brassica oleracea mitochondria. Brassica oleracea plants containing recombined mitochondria can possess unique phenotypic characteristics. Thus, protoplast fusion can provide plant breeders with a tool with which to create new Brassica oleracea plants having unique genotypic and phenotypic characteristics not present in nature. The diploid CMS Brassica oleracea plants contain a mitochondrion having DNA that has a restriction

fragment length polymorphism (RFLP) fingerprint as illustrated in Table 2 (fusion lines designation 930-1 and 998-5). Protoplast fusion techniques can be used to produce these CMS, cold tolerant, diploid Brassica oleracea plants comprising chloroplasts of fertile Brassica oleracea and a genotypically unique recombined mitochondrial genome. The mitochondria with DNA having the fingerprints as illustrated in Table 2 can also be bred into other Brassica oleracea plants, and thus into the seeds of those plants.

The present invention also provides a method for the preparation of cytoplasmic male sterile Brassica oleracea plants by protoplast fusion. The method includes isolating and fusing protoplasts from fertile Brassica oleracea plants with protoplasts from Brassica oleracea plants comprising Ogura CMS cytoplasm under conditions that cause formation of a recombined

mitochondrial genome; culturing the fused protoplasts; and generating plants from tissue derived from the fused protoplasts; wherein the recombined mitochondrial genome of the plants comprise genetic elements of both the fertile and Ogura CMS plant mitochondrial genome. In a particularly preferred embodiment, the method includes fusing protoplasts isolated from fertile B. oleracea var. italica (broccoli) plants with protoplasts isolated from enucleated B. oleracea var. italica (broccoli) plants comprising Ogura CMS

cytoplasm. DETAILED DESCRIPTION OF THE INVENTION

Protoplast fusion provides the means to recombine cytoplasmic elements of plant cells. This technique can be accomplished by a number of techniques.

According to the present invention, it is preferably accomplished by employing polyethylene glycol (PEG) in the presence of a fusion buffer to cause agglutination. The agglutinated cells are then diluted and washed with appropriate media. Such protoplast culture procedures may be effected under the media and conditions

disclosed in Kao, Cell Genetics in Higher Plants.

Dudets et al., eds., pp. 233-238 (1977) or Pelletier et al. Mol. Gen. Genet., 191, 244-250 (1983). In the present invention, the protoplast culture procedures and media used were based primarily on Pelletier's procedure with some modifications. These include changes in growth regulator composition and

concentration and changes in carbohydrate composition. The first step required to accomplish protoplast fusion is protoplast purification. Among the possible sources of pure protoplasts are plant leaves and suspension tissue cultures. Protoplasts can be obtained from whole leaves of Brassica oleracea plants by slicing the leaves and then treating the pieces with cell wall-degrading enzymes. Among the enzymes which are useful for this purpose are cellulases and pectinases.

Examples of commercially available protoplasting enzyme preparations include pectolyase Y23 and cellulase Onozuka RIO. Protoplasts can be obtained from

suspension tissue cultures using the same enzymes.

Specifically, protoplasts can be obtained from green plant material, e.g., leaf material, and/or from white plant material, e.g., etiolated seedlings, cell

suspension cultures, roots or bleached plant material, according to conventional methods such as the method disclosed by Glimelius, Physiologia Plantarum. 61. 38 (1984), for the regeneration of hypocotyl protoplasts.

The inactivated or nucleus-free protoplasts of an Ogura CMS Brassica oleracea plant are obtained from

corresponding Ogura CMS Brassica oleracea plant cells or protoplasts by irradiation nuclear inactivation or by standard methods known for the removal of the nucleus from cell material, such as centrifugation.

The inactivation of the nucleus by irradiation can be effected with the aid of gamma radiation, UV radiation, or X-rays, for example. Where irradiation is effected with an X-ray source, the appropriate X-ray dosage may, for example, be established by determining the minimum level of X-ray irradiation killing 100% of the

protoplast population. The percentage of dead cells is typically estimated by counting the number of formed colonies after 10-20 days in culture.

To obtain optimal conditions for the development of cell colonies at low density, it is desirable to use a feeder layer, a pre-conditioned culture medium, or an appropriate cell rescue procedure. Upon determination of the minimum dosage required for the inactivation of cell divisions, the protoplasts are preferably exposed to five increments of X-ray level: the minimum dosage, 10 and 20 krad above and below the minimum dosage.

Satisfactory nucleus inactivation in general can also be achieved by gamma irradiation with ⁶⁰Co at a dose of 3-30 krad. Nucleus elimination can also be carried out by incubation of protoplasts in high osmotic medium to obtain nucleus-free subprotoplasts. Protoplast fusion using the inactivated and/or nucleus-free protoplasts according to the invention is

conveniently effected by mixing approximately equal volumes of the two protoplasts to be fused.

Preferably, the protoplasts are mesophyll protoplasts and suspension cell-protoplasts. The protoplast fusion process can be monitored by ultraviolet microscopic observation. This is accomplished by staining the parental protoplasts with fluorescent dyes, e.g., fluorescein isothiocyanate. The nuclei of mesophyll protoplasts fluoresce red and the cytoplasm of

suspension cell protoplasts fluoresce yellow. Fused cells fluoresce both colors. The fusion process is generally carried out under conditions that cause diploid formation and prevent the formation of tetraploid products. Irradiation of one of the two protoplast preparations is used to inhibit tetraploid formation. Irradiation causes severe chromosomal damage; thus, irradiated cells are unable to replicate and, upon fusion, do not contribute viable chromosomes to the fusion protoplast. The

nonirradiated protoplast contributes a viable nucleus to the fusion product.

Specifically, in the method of the present invention, after about 3-5 minutes of fusing in a solution of PEG 8000, CaCl₂, 4% sucrose, and 10% DMSO, the mixture is diluted with a solution containing 50 mM glycine, 300 mM glucose, and 100 mM CaCl₂. The pH is preferably about 9.0. Fusing protoplasts are incubated for about 8 additional minutes and the protoplasts are

subsequently washed in wash media. The wash media includes 154 mM NaCl, 125 mM CaCl₂, 5 mM KCl, 5 mM glucose, 5 mM MES (2-[N-morpholino]ethanesulfonic acid), and is adjusted to a pH of 5.6. The protoplasts are suspended in the wash media and then collected by centrifugation (about 500 rpm for about 10 minutes) . The protoplast pellet is suspended in a rinsing

solution. The rinsing solution includes appropriate salts (e.g., KNO₃, CaCl₂, MgSO₄, KH₂PO₄), sucrose, 1.2% HEPES (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] and is adjusted to a pH of 5.6. The floating band of protoplasts are collected and then cultured. The obtained fusion products are then cultivated in an appropriate culture medium comprising a well-balanced nutrient supply for protoplast growth, containing micro- and macro-elements, vitamins, amino acids, and small amounts of carbohydrates, e.g., various sugars such as glucose. Glucose serves as a carbon source as well as an osmotic stabilizer. The culture medium comprises plant hormones, e.g., auxins and cytokinins, which are able to regulate cell division and shoot regeneration. Examples of suitable auxins are

naphthalene acetic acid (NAA),

2,4-dichlorophenoxyacetic acid (2,4-D), and indole-3-acetic acid (IAA). Examples of suitable cytokinins include benzyl aminopurine (BAP) and ZEATIN (6-[4-hydroxy-3-methylbut-2-enylamino]purine). In general NAA and 2,4-D are used in combination with BAP to initiate cell division. In this case, the ratio auxin/cytokinin must be high, e.g., greater than 1.

Specifically, in the method of the present invention, fusion protoplasts are cultured at a cell density of 5 × 10⁴ per mL in 0.4 M (carbohydrate concentration) modified Pelletier B liquid medium (see below for precise details of the media used herein). Osmotic concentration is preferably approximately 520 mOsM/kg H₂O as measured by an Advanced Wide-Range Osmometer 3WII manufactured by Advanced Instruments, Inc., Needham Heights, MA. The plates are cultured at 23-25°C in the dark for the first three days. After 7-10 days in culture, most of the protoplasts regenerate cell-walls, and a number of them show cell division. After adding modified Pelletier C liquid medium, the cells are cultured for another 3-5 days. At this stage, the surviving protoplasts produce multicellular clusters. The surviving cells are then diluted with modified Pelletier C liquid medium and transferred to quadrant plates using Pelletier D solid medium as the

"Reservoir" medium. Quadrant plates are set up by cutting slits (less than 1 mm wide) the length of the bottom of the ribs of Falcon quadrant plates.

Subsequently, molten Pelletier D solid medium is poured into 2 opposite quadrants and allowed to solidify.

Protoplasts in liquid media as described above are placed into the other two quadrants. This arrangement allows diffusion between the liquid and solid media during the course of protoplast culture. Typically, microcalli appear after 5-7 days of

culturing on the quadrant plates. During this time, the osmotic concentration of the liquid media is reduced as a result of solutes diffusing into the solid media. Gradually, the solutes of both media come to equilibrium. Because the growing of microcalli require lowered osmotic concentration, the quadrant plate system provides a means to gradually lower osmotic concentration in the media supporting the microcalli growth. After the microcalli appear, they are

transferred to Pelletier D solid medium and allowed to grow about one week. This medium promotes rapid callus proliferation.

Subsequently, the calli are transferred to modified Pelletier solid medium to induce shoot formation.

Shoots typically develop on this medium during a period of 2-3 weeks and typically appear as clusters of organized leafy shoot structures. These shoots are then allowed to grow and are transferred to rooting media. The mitochondrial DNA (mtDNA) of the obtained plantlets can then be characterized in a manner known in the art, such as, for example, employing suitable restriction endonucleases and comparing the thus obtained

mitochondrial DNA digest pattern of the fusion products with that of the parental lines. This is known as RFLP or restriction fragment length polymorphism, an

analytical technique used to detect genome polymorphism by comparing restriction maps of DNA from individuals. Variations in the position of restriction sites will result in different length restriction fragments which can be identified using southern blotting with suitable hybridization probes. Such DNA sequence variations display Mendelian inheritance and can be used as genetic markers in linkage studies.

Specifically, in the present invention, cloned

fragments of the CMS Ogura radish or Brassica

campestris mitochondrial DNA can serve as hybridization probes for DNA blot hybridizations. Techniques for the isolation of DNA can be found in Murray et al., Nucleic Acids Research. 8, 4321-4325 (1990) and Mettler et al., Plant Molecular Biology Reporter. 5, 346-348 (1987). Techniques for the analysis of organelle genomes in somatic hybrids derived from CMS Brassica oleracea and atrazine-resistant Brassica campestris can be found in Robertson et al., Theor. Appl . Genet .. 74, 1474-1478 (1987). Ogura CMS Brassica oleracea plants may be obtained by classical breeding techniques from Brassica oleracea and CMS Raphanus sativus as disclosed in

Bannerot et al., Proc. Ecarpia Meeting Cruciferae. 52- 54 (1974). It will be appreciated that the Brassica plants of the invention having the cold tolerant phenotype correlates with the substantial absence of the chloroplasts derived from Ogura CMS. These may be employed as starting material for the preparation of other Brassica oleracea varieties having the desired mitochondria of the Ogura CMS cytoplasm and chloroplasts of normal fertile Brassica oleracea and optionally additional desirable traits by in vi tro and/or crossing

techniques. Such in vi tro and crossing techniques are known in the art by the skilled breeder.

The invention will be further described by reference to the following detailed examples. All reagents are commercially available from standard sources, such as Sigma Chemical Co., St. Louis, MO. The Pelletier

Solutions listed below are described in Pelletier et al., Mol. Gen. Genet.. 191, 244-250 (1983). Biorganic and buffer salts used were standard research quality materials. Plant growth regulators and Tween 80 were cell culture tested. In general, methods used here are well-known to those skilled in the art.

EXAMPLE 1

A. Propogation of Broccoli Plants

A fertile Asgrow broccoli line BR206 and a sterile Asgrow broccoli line CMS BR362 (referred to in Tables l and 2 as "CMS") were used to produce protoplasts. Seeds of these lines were germinated and plants were grown at 23-25°C, under a 16-hour photoperiod with two 40-watt fluorescent bulbs for 25-35 days, then

transferred to a dark chamber for 2-3 days until leaf tissue turned yellowish.

B. Mesophyll Protoplast Isolation

Dark-treated, slightly yellowish, well-expanded young leaves of the fertile broccoli line BR206, about 4-5 cm in diameter, were used to isolate mesophyll

protoplasts. Leaves were surface sterilized by dipping in 70% ethanol briefly, then submerging the leaf-tissue in 10% Clorox containing Tween 20 for three minutes. The leaf-tissue was rinsed with sterile distilled water 5 times. Leaves were cut into 3 × 7 mm slices and suspended in the 1.8% Enzyme

Mixture overnight at room temperature (20-25°C).

Digested leaf-tissue was filtered through a double layer of cheesecloth to remove undigested debris. The filtrate was transferred into a narrow neck centrifuge tube, spun in an IEC clinical centrifuge at 500 revolutions per minute (rpm) for 10 minutes.

Mesophyll protoplasts floated as a band in the tube neck. The protoplast band was transferred to another narrow neck centrifuge tube in the Protoplast Washing Solution and spun at 500 rpm for 10 minutes in an IEC clinical centrifuge. The washed protoplasts formed a floating band. The washing procedure was repeated one more time. The purified mesophyll protoplasts were transferred to petri-dish ready for fusion.

C. Suspension Culture Establishment For callus initiation, leaf-tissue or hypocotyl of sterile broccoli line CMS BR 362 was the explant.

Pieces were cultured on callus medium [MS salt

supplemented with NAA (2 mg/L), 2,4-D (1 mg/L) and kinetin (1 mg/L)] for 3-5 weeks, and subcultured every 1-2 weeks. Two types of callus tissue (friable and compact type) were usually formed, but only the friable type was used to establish homogeneous

suspension cultures. The suspension culture medium used was the same as the callus medium except without agar. Fifty mL suspension cultures were established by transferring about 1-2 mL of friable callus into about 20 mL of liquid medium in a 125-mL flask. Flasks were maintained at 25°C, with a 16-hour

photoperiod under two 40-watt fluorescent tubes, shaking at 150 rpm. These suspensions were

subcultured every 8-10 days over a period of weeks to months until homogeneous cultures were established. These were maintained under the same culturing regime with the same subculturing schedule prior to use in protoplasting procedures. D. Suspension Protoplast Isolation and Irradiation

Suspension cells (e.g., cell clusters) in early to middle linear growth phase (5-7 days after

subculturing in fresh medium) were used as the CMS protoplast source. Carboxyfluorescein (0.1 mg/10 mL) was added to the suspension culture medium three days before protoplast isolation. Suspension cell-clusters were harvested by centrifugation at 1000 rpm; the cells were washed in the 0.6 M Suspension Protoplast Washing Solution.

Washed cells were transferred to a 2.0% Enzyme Mixture

(for suspension cell protoplast isolation). About 5-7 mL of enzyme mixture were used for each gram of washed suspension cells. These cells were suspended in this enzyme mixture at room temperature overnight in the dark. The macerated suspension-cell mixture was filtered through a double- layer of cheese cloth, and the lysate was transferred into a narrow-neck

centrifuge tube. The tube was spun at 1000 rpm for 10 minutes in an IEC clinical centrifuge; protoplasts floated to the top and formed a band in the tube neck.

The protoplast-band was placed in a 60 x 15 mm petri-dish preparation for X-ray irradiation treatment of protoplasts. X-ray irradiation treatment (20-25 krad) was carried out immediately after the

protoplasts were collected. The protoplasts were ready for fusion after being irradiated and washed twice in the 0.6 M Suspension Protoplast Washing

Solution.

E. Protoplast Fusion Procedure

A PEG-mediated fusion procedure was used. Equal volumes of mesophyll protoplasts and

suspension-cell-protoplasts, e.g., 0.5 mL each

(approximately 2 × 10⁶ protoplasts), were mixed. Then 1 mL of PEG Solution was added to the protoplast mixture and incubated at room temperature for 3 minutes. The fusion process was observed under

UV-microscope. The nuclei of mesophyll protoplasts fluoresced red, and the suspension-cell-protoplasts (entire cytoplasm) fluoresced bright yellow. Thus, fused cells were able to be identified unambiguously. After 3-5 minutes of fusing, the mixture was

immediately diluted with the High pH, High Calcium

Eluting Solution. After an additional 8 minutes, the protoplasts were washed with the W5 Solution by suspending the PEG treated protoplasts in the W5

Solution and subsequent centrifugation for 5 minutes at 500 rpm. The liquid was decanted, the

protoplast-pellet suspended in the Protoplast Rinsing Solution. The fusion-treated-protoplasts were washed one more time by centrifugation at 500 rpm for 10 minutes. The floating band of protoplasts were then ready for plating.

F. Protoplast Culture

Fusion-protoplasts were cultured at a cell density of 5 × 10⁴ per mL in modified 0.4 M liquid Pelletier B medium, which was modified by using NAA (0.1 mg/L), 2,4-D (1.0 mg/L), BA (0.5 mg/L), 0.4 M glucose, and no Tween 80 or D-mannitol instead of the concentrations for these components listed above for Pelletier B medium. Osmotic concentration was approximately 520 mOsM/kg H₂O. The plates were cultured at 23-25°C in the dark for the first three days. After 7-10 days of culturing, most of the protoplasts regenerated

cell -walls, and a number of them showed cell division. A half volume relative to the protoplast culture media volume of modified Pelletier C liquid medium (which contained NAA (1.0 mg/L), 2,4-D (0.25 mg/L), and BA (1.0 mg/L) instead of the concentrations listed above for these components) was added, and the cells were cultured for another 3-5 days. At this stage, the surviving protoplasts produced multicellular clusters. The surviving cells were diluted with 1-2 volumes of modified Pelletier C (same modifications as listed just above) liquid medium and transferred to quadrant plates (Falcon 1009X Plate Petri Dish) using Pelletier D solid medium as the "Reservoir" medium.

G. Microcalli Culture and Plant Regeneration

and Root Induction

Microcalli appeared after 5-7 days of culturing in the quadrant plates. The microcalli were transferred solid Pelletier D solid medium for a week. To induce shoot-regeneration, calli were transferred onto modified Pelletier E medium, which contained BA (1.5 mg/L), ZEATIN (1.0 mg/L), IAA (0.5 mg/L), GA₃ (0.1 mg/L) sucrose, (30,000 μ.g/1) and no D-Mannitol, instead of the concentrations for these components listed above. After 2-3 weeks, clusters of organized leafy shoot structures were harvested and transferred into modified Pelletier E medium, which contained kinetin (0.5 mg/L), IAA (0.25 mg/L), GA₃ (0.2 mg/L), sucrose (30,000 mg/L), and no D-mannitol instead of the concentrations of these components listed above. Shoots elongated on this medium. After they reached 1 cm in length, they were transferred to MS medium

(described in Murashige and Skoog, Plant Physiol., 15. 473-497 (1962)) containing IBA (1.0 mg/L) in 25 × 125 mm tubes. Once the plants were sufficiently large, they were potted into Jiffy7 plugs and placed in a mist chamber. Subsequently, they were "hardened-off" by methods known to those skilled in the art and transplanted into the field.

H. Evaluation of Sterility, Cold Tolerance.

and Seed Set

The plants were grown in the field to flowering. Male fertile and sterile plants were identified. Sterile plants were bee pollinated. Male sterile plants that displayed good seed set under field conditions were identified and seed harvested from them. Seed of these plants was germinated at 8°C in a controlled environment in magenta boxes in agar. After 2 weeks the seedlings were evaluated for color. Seedlings showing green color were grown in the greenhouse, and subjected to controlled pollinations. During the first two generations, seedlings were screened at 8°C for color to verify cold tolerance. Subsequently, novel CMS lines were used in a CMS conversion program. Routinely, for subsequent generations, broccoli seed and cabbage seed were germinated in the greenhouse. The resulting plants were then transplanted to the field approximately one month after seeding. Broccoli plants were selected for type, brought into the greenhouse and subjected to controlled pollinations. Cabbage plants were selected, stored at 2- 4° C for ten weeks, transferred to the greenhouse, and

pollinated. After 2 to 4 generations of backcrossing, selected CMS lines were placed in pollination cages in California with fertile pollinator lines. For CMS broccoli lines derived from 930-1 seed production ranged from 1.7 to 12.1 grams/plant, whereas for a fertile broccoli line, seed production ranged from 0.8 to 3.5 grams/plant. For the CMS cabbage lines derived from 998-5, seed production ranged from 0.6 to 16.3 grams/plant, whereas for a fertile cabbage line, seed production range from 0.5 to 3.2 grams/plant.

I. Total DNA Extraction From Fusion Plants

Total cellular DNA was extracted from progeny of two plants regenerated from fusion procedures. The regenerated fusion plants were designated PC8913998-5 and PC8919930-1. Regenerated fusion plants were produced in the laboratory; they were subsequently transplanted into the field and open pollinated.

Progeny derived from PC8913998-5 are designated 998-5 in this application; progeny derived from PC8919930-1 are designated 930-1 in this application.

Seed obtained from regenerated fusion plants was germinated and plants were grown in the greenhouse under usual conditions. DNA was extracted from young plants with fully expanded leaves (2-4 cm in width). Total cellular DNA was extracted by a CTAB extraction method. The 1% CTAB Extraction Buffer was prepared and preheated to 65°C for 5-10 minutes prior to use. The following was added to each mL of the CTAB

Extraction Buffer just before using: 10 μL of

2-mercaptoethanol, 6 μL of Ribonuclease T (5,000 U/mL) Sigma R-8251, and 25 μL of Ribonuclease A (10 mg/mL) Sigma R-4875. Three or four newly expanding leaves (0.5-1 gm fresh weight) were placed into the bottom corner of a ZIPLOC brand bag. One mL of preheated the 1% CTAB Extraction Buffer was added to the leaf sample. The ZIPLOC brand bag was placed flat on a hard surface. A one-liter Corning media-bottle was firmly rolled across the surface of the bag repeatedly until the leaf tissue was disrupted and had the consistency of applesauce. The macerated sample was moved to a bottom corner of the ZIPLOC brand bag and the corner was cut with a scissors. The entire sample was squeezed into a sterile 15 -mL Falcon tube and incubated at 70°C for 30 minutes. The sample was cooled for 5 minutes at room temperature (20-25°C). One mL of chloroform-octanol (24:1 v/v) was added, and the sample was vortexed for 1 second to mix thoroughly. The samples were then centrifuged in a Beckman GH 3.7 rotor (Beckman GPR centrifuge) at 2500 rpm at 25°C for 5 minutes to separate phases. The aqueous phase (about 1000 μL) was then transferred to a sterile 1.5-mL Eppendorf tube and 1.5 μL of RNAse Tl (10 mg/mL) was added. An equal volume of the 1% CTAB Extraction Buffer was added to each sample. The tube was inverted a few times and incubated at room temperature for 30

minutes.

The sample was centrifuged in an Eppendorf microfuge for 60 seconds to pelletize the precipitate. The supernatant was discarded, and the tube was inverted on a paper towel to drain. A high salt solution (500 μL of a solution of 10 mM Tris pH 8.0, 1 M NaCl, 1 mM EDTA pH 8.0) was added, and the sample was incubated at 65°C for 15 minutes to dissolve the DNA. One mL of 100% ethanol was added and the sample was placed at -20°C for one hour, or overnight if necessary, to precipitate DNA. DNA was hooked or spooled with a 1.5 mL capillary pipet and placed into a sterile 1.5-mL Eppendorf tube. The DNA pellet was washed by adding l mL of wash solution (80% ethanol, 15 mM ammonium acetate) and incubated at room temperature for 15 minutes. The washed DNA was dissolved in 300 μL of sterile water.

J. Restriction Digestion of Brassica oleracea DNA

Total DNA of progeny of Brassica oleracea fusion products was digested, electrophoresced, Southern blotted, and hybridized by standard techniques such as those disclosed in Sambrook et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press (1989). Specifically, total cellular DNA was digested as follows: DNA was added to 1.0-mL Skatron tubes; a bulk digestion mix was prepared by combining RNAse (10 mg/mL), 10 X Restriction Enzyme Reaction Buffer

(supplied with restriction enzymes by GIBCO BRL), 0.1 M Spermidine and the appropriate restriction enzyme, water was then added to bring the solution to final volume so that all reagents were all final lx

concentrations, and the resulting solution was placed on ice; this digestion mix was then aliquoted to each tube. Generally, 0.5-2.0 μg of DNA per lane was found to be adequate for detection of single copy sequences. The digestion mix was then incubated at 37°C for at least 4 hours. The reaction was then stopped by adding 10% ethanol to the digestion mix. The solution was then spun in a Technospin centrifuge at 4000 rpm for 40 minutes. The supernatant was poured off into a fresh set of Skatron tubes and the NaCl concentration was brought to 0.25 M by the addition of 5 M NaCl.

Into each tube was added ice cold ethanol (2.5 times the volume of the supernatant). These tubes were than placed at -20°C for 30 minutes, followed by

centrifugation at full-speed for 5 minutes just as the samples thawed. The supernatant was poured off and the pellet was washed with 175 μL ice cold 80%

ethanol. The pellets were briefly dried by inversion at room temperature followed by drying in a vacuum desiccator attached to a lyophilizer for 10-15 minutes (tubes placed upright) . The tubes were covered with Kleenex tissue before drying. The pellet was then dissolved in a volume of TE-8.0 (30 μL/5 μg DNA & 60 μL/10 μg). Generally, the pellets dissolved in 1-2 hours and stored overnight at 4°C in a sealed

refrigerator. Electrophoresis was carried out in neutral agarose gels. The mitochondrial genome in progeny of somatic hybrid plants then was analyzed by Southern blot hybridization analysis.

K. RFLP MtDNA Fingerprint

Southern blot hybridizations identified parental and non-parental mitochondrial DNA (mtDNA) restriction fragments in each of the fusion lines. Total cellular DNA was digested with seven different restriction endonucleases, DNA restriction fragments were

electrophoretically separated on agarose gels, and the digested DNA was blotted onto nylon membrane (HYBOND N, Amersham, Chicago, Illinois). Subsequently, the blots were hybridized with ³²P-labelled SalI fragments of Ogura CMS mitochondrial DNA or PstI fragments of Brassica campetris mitochondrial DNA. Autoradiography detected hybridization signals. An inventory of hybridization signals, which we designate as the mtDNA fingerprint, was generated for each parent and somatic hybrid. mtDNA fingerprint analysis for each somatic hybrid can clearly discriminate the present fusion products (somatic hybrids) and others characterized in the literature. This inventory of fragments, i.e., fingerprint, for each fusion line includes fragments shared by both parents, fragments unique to one or the other parent (restriction fragment length

polymorphisms), and nonparental or novel fragments. Specifically, cloned fragments of CMS Ogura radish (Makaroff and Palmer, Mol . Cell Biol., 8, 1474-1478, (1988)) or Brassica campestris (Palmer and Herbon, J. Mol. Evol., 28, 87-97, (1988)) mitochondrial DNA served as hybridization probes for DNA blot

hybridizations. Clones of Ogura and B. campestris mtDNA fragments were obtained from Dr. Chris Makaroff, Miami University, Department of Chemistry, Oxford, Ohio. Because Raphanus and Brassica mitochondrial sequences are conserved, both Ogura and Brassica campestris probes hybridized with Brassica oleracea mitochondrial sequences. Total cellular DNA extracted from fusion parents and progeny was digested with 7 different restriction enzymes: SalI, PstI, KpnI, NruI, NcoI, and BglI, and BglII. Following

electrophoresis, blotting, hybridization, and

autoradiography as described above, an invention of restriction fragments was generated for each fusion parent and somatic hybrid (Tables 1 and 2).

Restriction fragments identified in somatic hybrid parents and the two preferred progeny lines designated 930-1 and 998-5 by each enzyme probe pair are

presented in Table 1. For example, fragments

identified in SalI digests of total DNA probed with the mitochondrial DNA cloned fragment Ogura 1 (Ogl) are listed under the heading "SalI Ogl". This is the first group shown in Table 1. Other enzyme probe combinations tested are listed in the upper left of each group shown in Table 1. After each probe/enzyme combination, the cloned restriction fragment used as a probe is noted. The identity of the clone restriction fragment is indicated by size. These are shown in Figure 2 of Makaroff and Palmer (1988) for Ogura probes and Fig. 5 of Palmer and Herbon (1988) for Brassica campestris probes. The Ogura probes are SalI fragments and the Brassica campestris probes are PstI fragments.

Restriction fragments identified by each enzyme probe combination are indicated by size in each group. Sizes are indicated in the second line in each enzyme probe grouping. For example, the enzyme probe pair "Sail Ogl" detected six different restriction

fragments in the DNA samples tested: 33.5, 19.5, 15.4, 13.3, 11.8, and 3.7 kilobases. Presence or absence of these mtDNA fragments in plants shown for each enzyme probe pair is indicated by "1" or "0", respectively. For example, the 3.7 kilobase fragment detected in Sail digestions probed with Ogl probe (Sail Ogl) is absent in the CMS parent and present in BR206 fertile parent and present in the two somatic hybrid lines of the invention 930-1 and 998-5.

Each enzyme probe pair listed in Table 1 identified different sets of fragments in the two somatic hybrid parents. For example, in the second enzyme probe pair "SalI D23", the CMS parent shows a 4.4 kb band and not a 3.9 kb band, while the fertile parent (BR206) displays the 3.9 kb band and not the 4.4 kb band.

These polymorphisms are known as restriction fragment length polymorphisms (RFLPs). RFLPs clearly

distinguish the two somatic hybrid parents in Table 1.

Each enzyme probe pair identified a combination of RFLPs for each somatic hybrid and parent. For

example, the RFLP combination identified by the Sail Ogl enzyme probe pair in somatic hybrid lines 930-1 and 998-5 is designated "1.0". The RFLP pair

identified by the the SalI Og4 enzyme probe pair is designated "3.0" (Table 1). Each somatic hybrid line was characterized by an RFLP combination for each enzyme probe pair tested. Each unique RFLP

combination was assigned a number. These numbers are indicated under "RFLP comb." for each enzyme probe pair in Table 1. For each Brassica oleracea somatic hybrid tested with various enzyme probe pairs, a unique set of RFLP combinations was identified. The set of RFLP

combinations identified for each line of somatic hybrids is called the mtDNA fingerprint. Table 2 illustrates that the MtDNA RFLP combinations (mtDNA fingerprint) found in each fusion line differ from the fingerprints of the other noted fusion lines.

Inspection of RFLP combinations identified in each fusion line reveals that each line possesses a

different complement of RFLP combinations. Pair-wise comparisons show that each fusion line mtDNA

fingerprint differs from all of the others. In Table 2, CMS parental restriction fragment combinations are identified with a number followed by a "C"; fertile-parental restriction fragment combinations are

identified by an "F".

The data presented in Tables 1 and 2 indicate that protoplast fusion induced the random rearrangement of mitochondrial genomes in the CMS somatic hybrid

Brassica oleracea cytoplasms disclosed herein. Thus, protoplast fusion allows for the random production of recombined mitochondrial genomes having unique

genotypic characteristics. These randomly produced recombined mitochondria contain portions of the Ogura CMS mitochondria and the fertile Brassica oleracea mitochondria. Brassica oleracea plants containing these recombined mitochondria can possess unique phenotypic characteristics as well.

Seeds were deposited with the .American Type Culture Collection, Rockville, MD 20852, comprising

mitochondria having the DNA RFLP pattern of fusion line 930-1 with Accession No. 75961 on December 7, 1994. Seeds were deposited with the American Type Culture Collection, Rockville, MD 20852, comprising

mitochondria having the DNA RFLP pattern of fusion line 998-1 with Accession No. 75960 on December 7, 1994. The following is a brief description of the backcross procedure which was used to convert broccoli and cabbage lines to CMS. The purpose of this backcross (BC) program is to convert a male fertile line into a cytoplasmic male sterile (CMS) "line which is isogeneic to the recurrent parent and male sterile. The first step was to develop desirable CMS lines to use as CMS sources in these conversions. This was done through protoplast fusion at EPG. Two CMS lines, source 998.5 and source 930.1, were identified as having the best male sterility and female fertility. These two lines had relatively normal flower structure and set good quantities of seed when allowed to naturally pollinate in the field.

Starting with these CMS sources, promising broccoli and cabbage male fertile lines were selected to convert to CMS. Plants of the selected lines were brought into the greenhouse and pollen from the selected plants was transferred to the CMS donors by hand to produce seed. This first seed was F hybrid seed. This seed was then sown in the greenhouse in the case of broccoli and in the field in the case of cabbage. Selected CMS plants were then crossed back to the recurrent parent to produce the BC1 generation. For instance, if PC8919930.1 was originally crossed to CAB1, the hybrid (PCB919930.1×CAB1) was then

backcrossed to CAB1. The next generation, the

backcross 1 generation plant would be backcrossed again to the recurrent parent [ (PCB919930.1×CAB)×CAB] would backcrossed again to CAB1 to yield the BC2 generation. This is continued each generation in order to produce CMS lines virtually identical to the recurrent fertile lines. Each generation of backcrossing makes the CMS line closer and closer to the recurrent parent both

genotypically and phenotypically as follows: F₁ 50% of the genes in the CMS plant and the

recurrent parent are identical

BC1 75% of the genes are identical

BC2 87.6% of the genes are identical

BC3 93.76% of the genes are identical

BC4 96.875% of the genes are identical

With each succeeding backcross the remaining

difference between the backcross line and the

recurrent parent is halved.

The results of this backcross are shown in Table I for the 998.5 source, and Table II for the 930.1 source. The tables contain two columns: the first names the pedigree line derived from the 998.5 or 930.1 source; and the second specifies the backcross series.

The data establish proof that the CMS trait is stable and can be crossed into a variety of genetic

backgrounds using conventional breeding techniques to generate diversity in the CMS lines.

The entire disclosures of all publications, patents, and patent documents are incorporated herein by reference, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred

embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

WE CLAIM:

1. A cytoplasmic male sterile, cold tolerant, diploid Brassica oleracea plant comprising cells

comprising chloroplasts derived from a fertile

Brassica oleracea plant and a recombined

mitochondrial genome provided by Ogura CMS

mitochondria and said fertile Brassica oleracea plant.

2. The plant of claim 1 wherein the plant is

broccoli.

3. The plant of claim 2 where the plant comprises

mitochondria having DNA with a restriction

fragment length polymorphism fingerprint as illustrated in Table 2 under the designation 930- 1.

4. A Brassica oleracea seed from the plant according to claim 1.

5. The plant of claim 1 whereon the plant comprises mitochondrial genome having a restriction fragment length polymorphism fingerprint as illustrated in Table 2 under the designation 998-5.

6. The plant of claim 5 wherein the plant is cabbage.

7. A Brassica oleracea mitochondrion comprising a DNA having a restriction fragment length polymorphism fingerprint as illustrated in Table 2 under the designation 998-5.

8. A Brassica oleracea plant comprising the

mitochondrion of claim 8.

9. A Brassica oleracea seed comprising the

mitochondrion of claim 8.

10. A method for the preparation of cytoplasmic male sterile Brassica oleracea plants comprising:

(a) isolating protoplasts from fertile Brassica oleracea plants and protoplasts from Brassica oleracea plants comprising Ogura CMS

cytoplasm;

(b) fusing the protoplasts isolated from fertile Brassica oleracea plants with the protoplasts isolated from Brassica oleracea plants comprising Ogura CMS cytoplasm under

conditions that cause formation of a

recombined mitochondrial genome;

(c) culturing the fused protoplasts; and

(d) generating plants from tissue derived from the fused protoplasts;

wherein the recombined mitochondrial genome of the plants comprise genetic elements of both the fertile and Ogura CMS plant mitochondrial genome.

11. The method of claim 11 wherein the step of

isolating comprises isolating protoplasts from fertile B. oleracea var. italica (broccoli) plants and protoplasts from B. oleracea var. italica (broccoli) plants comprising Ogura CMS cytoplasm.